Specific binding assays enable the detection of a particular analyte in a test sample based on the specific binding interaction between the analyte and an appropriate binding partner. The binding between the analyte and its binding partner is most often measured by use of a labeled reagent which becomes bound to the analyte, the binding partner, or some other intermediary binding substance as a function of the presence or amount of analyte in the test sample. Examples are where analyte is detected by binding of a labeled form of the binding partner or where analyte competes with a labeled form of the analyte itself or an analog thereof for binding to a binding partner. A common binding interaction between the analyte and binding partner is the immunochemical binding of an antigen or hapten to a corresponding antibody, although other binding phenomena such as hormone/receptor, drug/receptor, and like interactions as well as nucleic acid hybridization can be employed
Assays of this type are well known to be susceptible to a wide range of interferences potentially affecting any one of a number of performance parameters such as precision, sensitivity, specificity, and the like. Oftentimes the interferences are due to substances present in the test sample which cannot be controlled. Minimizing sample interference effects can be particularly vexing since the causative agent may not be evident nor easily determinable. Presented with a specific binding assay exhibiting an unacceptable sample interference, the skilled worker in many cases has no assurance that the interference can be significantly reduced, much less eliminated, by any predetermined modification of the assay procedure or reagent ingredients.
A number of approaches are known for attempting to reduce or minimize sample interferences in specific binding assays. For example, in order to eliminate nonspecific label (analyte-fluorescein) binding to endogeneous serum proteins in the fluorescence polarization immunoassay (FPIA) for digoxin, serum samples are treated with trichloroacetic acid (TCA) to remove all serum proteins prior to analysis. Similarly, endogenous serum fluorescence [which has been shown to interfere with the detection of digoxin by the fluorescence energy transfer immunoassay (FETI)] can be eliminated by pretreatment of the sample with a strong oxidizing agent. Clearly, interferences are not unique to homogeneous immunoassays but have also been observed in heterogeneous assay systems. For example, nonspecific binding of enzyme labeled antibodies to polystyrene tubes or plates in enzyme linked immunosorbent assays (ELISAs). This type of interference has been eliminated by the use of detergents and/or proteins (e.g., bovine serum albumin) in the pretreatment of ELISA plates or tubes or by inclusion of these substances in the enzyme antibody label buffer.
In many specific binding assays, the amount of labeled reagent present in the assay mixture must be known or fixed in order for precise measurement of the analyte to be accomplished. The labeled reagent, as is known, comprises a label portion, which provides the detectable response by which the analyte is determined, and a specific binding portion which participates in the various binding interactions upon which the principle of the assay is based. Where the amount of labeled reagent in the reaction mixture is critical, the presence of substances in the test sample which will effectively alter the availability of the labeled reagent either to produce its detectable response or participate in the necessary binding interactions, can substantially affect the precision of the assay performance. This can be particularly acute where the analyte is present in relatively low concentrations in the test sample and assay performance must be quite sensitive.
Reduction of non-specific binding of a fluorescer-labeled antibody to a slide in immunofluorescence tissue staining methods by addition of a structural analog having no fluorescence or low level fluorescence of a different wavelength is disclosed in European Patent Application No. 140,602. The presence of FAD-binding proteins in human blood is disclosed by Farhangi and Osserman, New Engl. J. Med. 294:177-183(1976) and Merrill et al, Recent Findings Concerning Mammalian Riboflavin-Binding Proteins, in Flavins and Flavoproteins, Massey and Williams ed., Elsevier/North Holland (New York 1982) pp. 508-513. Armeta et al, Anal. Biochem. 146:211-219(1985) describe the use of inactive .beta.-galactosidase to adsorb and inactivate a serum inhibitory factor in an enzyme immunoassay using .beta.-galactosidase as the label.
The present invention is intended to provide means for controlling sample interferences in specific binding assays which interferences are suspected or found by investigation to be due to the presence of substances in the sample that interact with the label portion of the labeled reagent.